The biosynthesis of terpenoids (isoprenoids) begins with the synthesis of geranyl diphosphate (GPP; C10), farnesyl diphosphate (FPP; C15) and geranylgeranyl diphosphate (GGPP; C20), which are straight-chain prenyl diphosphates, through the sequential condensation reactions of isopentenyl diphosphate (IPP; C5) with an allylic diphosphate substrate (FIG. 1). In FIG. 1, the abbreviations and words in boxes represent enzymes. Specifically, hmgR represents hydroxymethylglutaryl-CoA (HMG-CoA) reductase; GGPS represents GGPP synthase; and FPS represents FPP synthase.
Among prenyl diphosphates, FPP is the most significant biosynthetic intermediate. It is a precusor for the synthesis of tremendous kinds of terpenoids, e.g. steroids including ergosterol (provitamin D2), the side chains of quinone (vitamin K; VK), sesquiterpenes, squalene (SQ), the anchor molecules of farnesylated proteins, dolichols, bactoprenol, and natural rubber.
GGPP is also a biosynthetic intermediate in vivo, and is essential for the biosynthesis of such compounds as phytoene, lycopene, ficaprenol, retinol (vitamin A; VA), β-carotene (provitamin A), phylloquinone (vitamin K1; VK1), tocopherols (vitamin E; VE), the anchor molecules of geranylgeranylated proteins, the side chain of chlorophyll, gibberellins, and the ether lipid of archaea.
It is known that these prenyl diphosphates with up to 20 carbon atoms are condensed into trans forms ((E) forms) and are present as (E,E)-FPP and (E,E,E)-GGPP. Compounds with physiological activities are synthesized from these prenyl diphosphates or prenyl groups with up to 15 or 20 carbon atoms having the all trans (all-E) geometrical isomerism as precursors (K. Ogura and T. Koyama, (1998) Chemical Reviews, 98, 1263-1276; IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) Prenol Nomenclature, Recommendations 1986, (http://www.chem.qmw.ac.uk/iupac/misc/prenol.html)). Among prenyl diphosphates with up to 20 or 15 carbon atoms, the only one exception that has the cis ((Z) form) geometrical isomerism is neryl diphosphate (C10) known as a precursor for those monoterpenoids as represented by nerol. It has not yet been elucidated whether neryl diphosphate is synthesized through condensation of IPP with dimethylallyl diphosphate (DMAPP; 3,3-dimethylallyl diphosphate) as an allylic diphosphate substrate; or through isomerization of geranyl diphosphate (GPP) that is a trans ((E) form) geometrical isomer with 10 carbon atoms. Those isoprenoids that are synthesized through condensation into cis forms ((Z) forms), e.g. dolichols, bactoprenol (undecaprenol) or natural rubber, are also synthesized from (E,E)-FPP or (E,E,E)-GGPP as an allylic primer substrate (K. Ogura and T. Koyama, (1998) Chemical Reviews, 98, 1263-1276; IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) Prenol Nomenclature, Recommendations 1986, (http://www.chem.qmw.ac.uk/iupac/misc/prenol.html)).
(E,E)-Farnesol (FOH; C15), which is an alcohol derivative of (E,E)-FPP; (E)-nerolidol (NOH; C15), which is an isomer of the tertiary alcohol thereof; (E,E,E)-geranylgeraniol ((E,E,E)-GGOH; C20), which is an alcohol derivative of (E,E,E)-GGPP; and the like are known as fragrant substances in essential oils used as the ingredient of perfumes. FOH, NOH and (E,E,E)-GGPP are also important as starting materials for the synthesis of various compounds (including the above-mentioned vitamins) useful as pharmacological agents (FIG. 1).
Although it had been believed that all the biosynthesis of EPP is performed via the mevalonate pathway (a pathway in which IPP is synthesized from acetyl-CoA through mevalonate acid), M. Rohmer et al. elucidated a novel IPP biosynthetic pathway using bacteria at the end of 1980's. This is called the non-mevalonate pathway, DXP (1-deoxy-D-xylulose 5-phosphate) pathway, MEP (2-C-methyl-D-erythritol 4-phosphate) pathway or Rohmer pathway, in which IPP is synthesized from glyceraldehyde-3-phosphate and pyruvate through 1-deoxy-D-xylulose 5-phosphate. Thus, two major pathways, i.e. the mevalonate pathway and the non-mevalonate pathway, are known at present as synthetic pathways for IPP.
FOH and NOH are currently produced by chemical synthesis except for small amounts of them prepared from natural products such as essential oils. GGOH is also produced by chemical synthesis (Japanese Unexamined Patent Publication No. 8-13999). Chemically synthesized FOH, NOH or GOOH generally has the same carbon skeleton, but they are obtained as mixtures containing (E) (trans) double bond and (Z) (cis) double bond geometry. (E,E)-FOH, (E)-NOH or (E,E,E)-GGOH, each of which is (all-E) type, is a compound with the geometrical isomerism synthesized in biosynthetic pathways and is industrially valuable. In order to obtain (E,E)-FOH, (E)-NOH or (E,E,E)-GGOH in a pure form, refining by column chromatography, high precision distillation, etc. is necessary. However, it is difficult to carry out high precision distillation of FOH, a thermolable allyl alcohol, or its isomer FOH, or GGOH. Also, the refining of these substances by column chromatography is not suitable in industrial practice since it requires large quantities of solvent and column packings as well as complicated operations of analyzing and recovering serially eluting fractions and removing the solvent; thus, this method is complicated and requires high cost. Actually, the (E,E)-FOH and (E,E,E)-GGOH sold as experimental reagents are very expensive. Under circumstances, it is desired to establish a system for synthesizing not mixtures of cis- and trans-((Z)- and (E)-) isomers but pure products of (E,E)-FOH (hereinafter, just referred to as “FOH”), (E)-NOH (hereinafter, just referred to as “NHO”) and (E,E,E)-GGOH hereinafter, just referred to as “GGOH”) (i.e., the so-called active prenyl alcohols) in large quantities by controlling the generation of (E)- and (Z)-geometrical isomers or by utilizing the characteristics of repeat structures of reaction products.
The substrates for the biosynthesis of FOH, NOH or GGOH are provided via the mevalonate pathway in cells of, for example, Saccharomyces cerevisiae, a budding yeast. However, even when HMG-CoA reductase that is believed to be a key enzyme for the biosynthesis was used, it has been only discovered that the use increases the accumulation of squalene, a substance commercially available at a greatly low price than FOH and GGOH (Japanese Unexamined Patent Publication No. 5-192184; Donald et al., (1997) Appl. Environ. Microbiol. 63, 3341-3344). Further, it is known that 1.3 mg of FOH per liter of culture broth is accumulated when a squalene synthase gene-deficient clone (ATCC64031) was created by introducing mutations into the squalene synthase gene ERG9 of a particular budding yeast that had acquired sterol intake ability, and cultured (Chambon et al., (1990) Curr. Genet. 18, 41-46). The present inventor determined the nucleotide sequence of the squalene synthase gene ERG9 in ATCC64031, and confirmed that this clone has become a squalene synthase gene-deficient clone (erg9 clone) as a result of the introduction of substitution mutations into the coding region of ERG9 and thus acquired the productivity of 1.3 mg/L of FOH. In the coding region of the squalene synthase gene of ATCC64031, the nucleotide at position 745 has been changed from C to T and the nucleotide at position 797 from T to G. As a result, the amino acid residue at position 249 has changed from Gln to termination codon (Q249STOP) and the amino acid residue at position 266 from Ile to Arg (I266R) in the polypeptide encoded by this gene. These changes have led to the expression of a mutant squalene synthase in which the amino acid residue at position 249 and thereafter are deleted, and which has no enzyme activity. However, when ERG9 is made deficient in a conventional strain, the deficiency is lethal to the strain because the strain cannot synthesize ergosterol essential for growth and it has no function to intake sterols from the outside under conventional culture conditions. Thus, an ERG9-deficient clone cannot be obtained; it is impossible to construct an FOH production system. Furthermore, no method of biosynthesis of (E)-NOH (hereinafter, referred to as “NOH”) is known. Even if it is possible to provide to a ERG9-deficient clone with a novel character that avoids the lethality, the cultivation of such a clone will require disadvantageous conditions to industrial production, e.g., necessity to add ergosterol to the medium.
With respect to the biosynthesis of GGOH, production of 0.66-3.25 mg per liter of culture broth is achieved by culturing plant cells in Japanese Unexamined Patent Publication No. 9-238692. However, this method needs an expensive plant cell culture medium inappropriate for industrial application and also requires light for culturing cells. Thus, this method is not practical even when compared to the conventional GGOH preparation from natural products such as essential oils. There is known no method of biosynthesis of GGOH suitable for industrialization, e.g. biosynthesis by microorganisms.